1. Field of the Invention
This invention relates to a method for the preparation of an immune globulin solution suitable for intravenous use.
2. Description of the Prior Art
Immune globulin solutions contain antibodies in the same, or a higher, concentration as the starting product, the animal or human blood. They have been in use for 27 years with greater or lesser success in two versions in passive immune prophylaxy, for agamma-globulinaemias and other antibody deficient states and for infectious illnesses. The one version is an intramuscularly (or subcutaneously) applicable solution with a 160 g/l protein concentration and the other an intravenously applicable solution usually with a protein concentration of 50 g/l.
Both the immune globulin intramuscular (i.m.) preparations and also the immune globulin intraveneous (i.v.) preparations can vary considerably according to the method of production and suffer from the disadvantages that are to be found in their differing compatability and therapeutic effect.
Thus, for example, small volumes of a conventional immune globulin i.m. preparation can produce at the injection location painful irritations, which at higher doses become agony for the patient. Another drawback of these preparations is the fact that about one-third of the injected quantity of antibodies is proteolytically broken down, and can therefore develop no further therapeutic action and the effection portion is transported away from the muscular deposition place only relatively slowly, that is too slowly in the case of acute sickness. Since the immune globulin at present commercially available, when used for i.v. application has been found to lead to serious to life threatening anaphylactoid reactions, it has been subjected by the manufacturers of such preparations, when intended for i.v. use, to special physical and chemical treatment methods.
The immune globulin i.v. preparations developed in this way and at present commercially available exhibit, however, other production-related disadvantages, which can likewise be seen in general incompatability reactions, which increase drastically as the dose increases, and in too low and/or one-sided therapeutic effects. The harmful effects with i.v. application can extend, as secondary reaction, from a reddening of the face through a feeling of anxiety, headache, visual disturbances, nausea, vomiting, high temperature, shivering to circulatory failure with loss of consciousness. If, however, the compatability can be termed good, the corresponding immune globulins i.v. preparations are too weak or too one-sided in the context both of antibacterial and antiviral and antitoxic effect, or their dwell time in the patient organism is too short, that is their half-life is clearly less than 18-22 days, the value for intact antibodies of species IgG (see Barandun et al, Schweiz. med. Wschr. 106 (1976) 533-542). As an evaluation scale for the secondary reactions occurring in vivo, an invitro test has proved successful in addition to the known toxicity and pyrogenity tests in animal experiments and the clinical compatability test. This so-called complement forming reaction test (.dbd.KBR--Test; e.g., method of G. J. Stein and Van Ngu, J. Immunol., 65 (1950) 17-37) permits quantitative determination of substances in immune globulin preparations, which nonspecifically bind the complement. The assumption is adopted today that an immune globulin i.v. preparation with a Lysehemm value of &gt;50% where 2 C.H50 units are used (see Kabat and Mayer's Experimental Immunochemistry, 2nd Ed., 4th Printing, Charles C. Thomas Publisher, U.S.A.) in a KBR formula comprising 12.5 g/l immune globulin protein content, will to a high degree of probability exhibit individually the above-listed secondary reactions, while preparations with L.H. values &lt;50% very drastically limit the risk of dangerous secondary reactions. In the production of immune globulins i.v. preparations, methods have been used which in principle differ from one another; these should make it clear that the new method to be put forward here is distinguished from those already known.
Thus, for example, it is known that in order to produce an immune globulin preparation, the gamma globulin fraction of the serum, obtained in the usual way by ammonium sulphate precipitation or alcohol precipitation, may be treated at a pH value of 1.5 to 5.5 at a temperature between 0.degree. and 50.degree. C. for 2 hours to 2 days with pepsin, for example with 25,000 to 200,000 units of pepsin/100 g of protein to be broken down, with continual monitoring of the anticomplementary effect until the non-specific complement fixation is eliminated; the thus produced gamma globulin is then fractionated by precipitation with neutral salts or is ultrafiltered, separated from low-molecular separation products, sterilely filtered and if necessary freeze-dried (DT-Pat. No. 11 48 037).
The disadvantage of this method is that essentially only broken down protein molecules are obtained and that only a few antibodies remain in native form, so that the antibacterial and antiviral effectiveness is very low. Furthermore, it must be taken into account that on the one hand a complete proteolytic elimination of the non-specific complement binding is practically impossible to carry out since native immune globulins are also always broken down, so that the immunological effectiveness of the preparation is questionable. It is also necessary to inactivate the enzymes (e.g., pepsin or plasmin), since otherwise they will continue their proteolytic action during the storage of the preparation.
Attempts have also been made to use polyethylene glycol for the fractionation of blood plasma and for the precipitation of a purified antibody fraction from highly diluted aqueous solutions, but here the existence of non-specifically complement binding substances has not been verified (Chun et al., Anal. Biochem 19 (1967) 481-497).
It is furthermore known to produce an immune globulin preparation, which essentially is free of anticomplementarily active constituents, from a natural antibody-containing starting material, especially plasma, by fractionation and selective precipitation of the disturbing substances and possibly also precipitation of haepatitis virus or antigen, freeing the immune globulin from undesired proteins, after the actual fractionation of the starting material, separating complement-fixing substances from an aqueous solution so that the immune globulin itself remains in solution and adding at least one dissolving-inhibiting agent, namely a water-soluble salt and/or a linear, chainshaped, non-ionic polymer compound, the latter being used in a concentration, the dissolving-inhibiting action of which is essentially the same as that of 4.7-7 g/100 ml of polyethylene glycol with a molecular mass of between 4000 and 8000 Dalton units, whereby the aqueous solution in the latter case contains at most 4 g/100 ml protein (DT-OS No. 22 34 069).
It is furthermore known to produce an intravenously applicable immune globulin preparation by first treating an aqueous solution of a native immune globulin having a weakly alkaline pH with dithiothreitol in a concentration of less than 0.01 mol/liter and a mol ratio to the protein of 2.5 to 50 in the reaction mixture until about 2 to 4 disulphide bonds per globulin molecule are reduced, whereupon an aqueous solution of this reduced product at weakly alkaline pH is alkalized with at least 1 mol-equivalent, referred to dithiothreitol, of iodine acetamide and then the thus obtained, chemically modified immune globulin is separated from the non-protein-containing reaction products and the excess of reaction partners (DT-OS No. 23 11 333). in spite of numerous further investigations and proposals for obtaining an intraveneously usable immune globulin preparation, the products obtained have not so far proved satisfactory, because they either still possess too high Lysehemm values and/or too short a dwell time in the organism or a too low antibacterial, antiviral and antitoxic action so that a purposeful dosing of the preparation is adversely influenced.
It is an object of the present invention to provide a method of producing an intravenously usable immune globulin which does not suffer from the disadvantages of the known preparations or possesses them only in a medically and clinically tolerable extent and which therefore is distinguished by a no longer detectable or only very slight non-specific complement fixing tendency and, by a good antibacterial, antiviral and antitoxic action, depending upon how intensively and how frequently the proposed method is used in the preparation of a product unit.
The method of the present invention can always be used with success when immune globulin fractions containing unacceptable proportions of proteins having an anticomplementary effect need to have these proteins separated from them.